Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis

Abstract

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 μm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-β transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-β-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-β transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

Keywords: amphiregulin; connective tissue growth factor (CTGF); cytokine; fibrosis; pulmonary fibrosis; siRNA.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Physicochemical properties of the SAMiRNA.A, schematic of SAMiRNA nanoparticles. B, a representative cryo-transmission electron microscopy image of SAMiRNA nanoparticles. Shown is one representative of a minimum of three independent experiments. Scale bar = 100 nm. C and D, size distribution and PDI of AR and CTGF SAMiRNAs measured by Nano-Zetasizer. d, diameter. E, long-term stability of AR and CTGF SAMiRNAs measured by nanoparticle size and PDI. The data in C and E represent mean ± S.E. of evaluations with a minimum of four independent experiments. D shows one representative plot of six replicates.

FIGURE 2.

Silencing efficacy and nonspecific immune stimulatory effects of naked siRNAs and SAMiRNAs.A and B, NIH3T3 cells were treated with AR and CTGF naked siRNAs and SAMiRNAs, and then the expression of AR and CTGF mRNA was measured by real-time RT-PCR and compared with scrambled (Sc) siRNAs or SAMiRNAs, respectively. C, the expression of nonspecific innate immune cytokine was evaluated using mouse PBMCs stimulated by naked siRNAs or SAMiRNAs of AR (siAR), CTGF (siCTGF), β-galactosidase (si-β-gal), and their control siRNA (si-Cont) or SAMiRNA (SAMiRNA-Cont). 5 × 105 mouse PBMCs were seeded onto a 48-well plate and treated with PBS, 5 μm naked siRNAs, 1 or 5 μm lyophilized SAMiRNAs, 1 μm of encapsulated liposomes (SNALPs), Lipofectamine RNAimax only, and concanavalin A (ConA, 20 μg/ml). After 24-h incubation, cell culture supernatants were harvested, and the levels of representative innate immune cytokines (TNF-α, MCP-1, IFN-γ, IL-12(p70), and IL-6) were measured by luminex multiplex screening assay. Data represent mean ± S.E. of a minimum of three separate evaluations with duplicates.

FIGURE 3.

In vivo biodistribution of SAMiRNA in bleomycin and TGF-β Tg animal models of fibrosis.A, left panel, time kinetic ex vivo fluorescence images after intratracheal instillation of Cy5.5-labeled SAMiRNA in bleomycin (BLM)-induced fibrosis and TGF-β Tg mice. Right panel, quantitative kinetic evaluation of fluorescence intensity by measuring region of interest (ROI) values. B, left panel, time kinetic ex vivo fluorescence images after intravenous injection of Cy5.5-labeled SAMiRNA nanoparticles in WT, bleomycin-challenged, and TGF-β Tg mice. Right panel, quantitative kinetic evaluation of fluorescence intensity by measuring region of interest values. C, representative flow cytometric analysis on the SAMiRNA-targeted cells in the lung. 24 h after intratracheal injection of 3 mg/kg APC-Cy5.5-labeled SAMiRNAs into TGF-β Tg mice, lung cells were isolated and subjected to flow cytometric evaluation using FITC-labeled cell specific markers: Clara cell 10Kd (CC10) for airway epithelial cells, surfactant protein C (SPC) for alveolar epithelial cells, CD140a (mesenchymal cells, including fibroblasts and myofibroblasts), CD68 (macrophages), and CD3 (T cells). At least 10,000 cells/sample were analyzed. The percentages of Cy5.5-positive (+) SAMiRNA-targeted cells in each group of cells defined by cell surface marker are represented separately (n = 4 each, right panel). A and B, the ex vivo fluorescence images are representative of a minimum of 4 mice/group. The region of interest quantitation represents mean ± S.E. of evaluations with a minimum of four mice.

FIGURE 4.

Pharmacokinetic evaluation of SAMiRNA. Serum levels of AR siRNA were evaluated by quantitative RT-PCR at the indicated time points after intratracheal (i.t.) or intravenous (i.v.) delivery of AR SAMiRNAs (SAMiRNA-AR) (5 mg/kg). A, pharmacokinetic evaluation of SAMiRNA-AR in bleomycin-challenged mice. conc, concentration; ITI, intratracheal injection; AUC, area under the curve; Cmax, maximal concentration; Tmax, time to reach maximal concentration. B, pharmacokinetics of SAMiRNA-AR in TGF-β Tg mice. Each panel is a representative plot of a minimum of three independent evaluations.

FIGURE 5.

Time kinetic changes in the expression of CTGF, AR, and fibrosis-related genes and collagen deposition in bleomycin-challenged lungs.A–E, time kinetics in the expression of CTGF, AR, collagen 3A1, fibronectin, and elastin in the lungs of mice exposed to intratracheal bleomycin (BLM). F, kinetic changes of soluble collagen protein deposition in lungs as measured by Sircol assay. Data represent mean ± S.E. of a minimum of four mice treated with bleomycin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

In vivo silencing of AR and CTGF expression in a bleomycin model of pulmonary fibrosis. 8-week-old WT mice were challenged with bleomycin (BLM), and AR and CTGF SAMiRNAs (3 mg/kg) were delivered via intratracheal (i.t.) injection on days 9 and 12 after bleomycin challenge, and the mice were sacrificed and evaluated on day 14. A, schematic of the protocol used in this evaluation. B, the silencing effects on the expression of AR, CTGF, and genes associated with fibrosis evaluated by quantitative RT-PCR. Data are mean ± S.E. of a minimum of 5 mice/group. Cont, control. C, H&E stains of lungs from mice treated with vehicle (PBS), bleomycin, bleomycin and control (BLM+SAM-Con), AR (BLM-SAM-AR), and CTGF (BLM-SAM-CTGF) SAMiRNAs. Shown is a representative of a minimum of 5 mice/group. D, representative Mallory trichrome staining of the lungs of the mice in C. ##, p < 0.01; ###, p < 0.001 compared with vehicle (PBS) control; **, p < 0.01; ***, p < 0.001 compared with bleomycin-challenged mice treated with control SAMiRNA. Scale bars = 400 μm.

FIGURE 7.

In vivo silencing of AR and CTGF expression in a bleomycin model of pulmonary fibrosis by SAMiRNAs via intravenous delivery. 8-week-old WT mice were challenged with bleomycin (BLM) and treated with control, AR, and CTGF SAMiRNAs (3 mg/kg) delivered via intravenous injection on days 7, 9, and 11 after bleomycin challenge. The mice were sacrificed and evaluated on day 14. A, collagen content in the lungs from these mice was evaluated by Sircol collagen assay. Data are mean ± S.E. ##, p < 0.01 compared with PBS control; *, p < 0.05 compared with BLM-SAM-Cont. B, Mallory trichrome staining of lungs treated with vehicle (PBS), bleomycin, bleomycin and control (BLM+SAM-Cont), AR (BLM+SAM-AR), and CTGF (BLM+SAM-CTGF) SAMiRNA and CTGF+AR in combination (BLM+SAM-Combo). Scale bars = 400 μm.

FIGURE 8.

In vivo silencing of AR and CTGF SAMiRNAs significantly suppresses TGF-β-stimulated collagen accumulation in the lung. 8-week-old WT and TGF-β Tg mice were treated with control or AR and CTGF and SAMiRNAs alone or together via intravenous (i.v.) injection (5 mg/kg/mouse) on days 7, 9, and 11 after transgene induction with doxycycline (Dox). A, schematic of the protocol used in this evaluation. B, the silencing effects on mRNA expression of AR, CTGF, and genes associated with fibrosis were evaluated by quantitative RT-PCR. C, total collagen quantitation in the lung by Sircol assay. D, representative Mallory trichrome staining of lungs from WT and TGF-β Tg mice challenged with control (SAM-Cont), AR (SAM-AR), CTGF (SAM-CTGF), and AR + CTGF (SAM-Combo) SAMiRNAs. Data in B and C represent the mean ± S.E. of a minimum of 5 mice/group. D shows a representative of a minimum of 5 mice/group. ##, p < 0.01; *, p < 0.05, **, p < 0.01 compared with TGF-β Tg mice treated with control SAMiRNA.

FIGURE 9.

In vivo silencing of AR and CTGF SAMiRNAs restores the lung function of TGF-β Tg mice. 8-week-old WT and TGF-β Tg mice were treated with control or AR and CTGF SAMiRNAs alone or together via intravenous injection (5 mg/kg/mouse) on days 7, 9, and 11 after transgene induction with doxycycline. Lung function was measured by the flexiVent system. A, respiratory system resistance (Rrs). B, respiratory system elasticity (Ers). C, central airway resistance (Rn). D, respiratory system compliance (Crs). Data are mean ± S.E. of a minimum of 5 mice/group. #, p < 0.05; ##, p < 0.01; *, p < 0.05; **, p < 0.01 compared with TGF-β Tg mice treated with control SAMiRNA (SAM-Cont).